Pharmacologically effective substance and process for preparing it

ABSTRACT

A PHARMACOLOGICALLY ACTIVE SUBSTANCE ISOLATED FROM THE BARK OF RAVENSARA AROMATICA BY EXTRACTION OF THE BARK WITH A LOWER ALCOHOL, EVAPORATION OF THE EXTRACT TO GIVE A SOLID RESIDUE, AND CHROMATOGRAPHY OF THE RESIDUE.

April 11, 1972 GROEBEL ETAL PHARMACOLOGICALLY EFFECTIVE SUBSTANCE ANDPROCESS FOR PREPARING IT Filed Aug. 11, 1970 00m 00 00w 00w Ooo OON O0!O02 O02 000m 00mm 000m 00o o i 8 w w 5 2 w W 5: a v w T, 3 c w m Q \I\ Tq 8 l 9v on ON 2 N O m m N m m Q m mN Int. Cl. Asik 27/14 U.S. Cl.424-195 5 Claims ABSTRACT OF THE DISCLOSURE A pharmacologically activesubstance isolated from the bark of Ravensara aromatica by extraction ofthe bark with a lower alcohol, evaporation of the extract to give asolid residue, and chromatography of the residue.

This is a continuation-in-part of U.S. patent application Ser. No.731,425, filed May 23, 1968, now abandoned.

The present invention relates to a pharmacologically effective substanceand to a process for isolating it.

We have found that a pharmacologically highly effective substance havinga strong and long lasting blood pressure lowering activity can beisolated from Ravensara aromatica.

Ravensara aromatia is a plant belonging to the family Lauraceae which isfound in Madagascar and is also known by the scientific names of Laurusaromatic-a and Agatophyllum ravensara.

The new substance is isolated from the bark of Raven:- sarc aromatz'ca,which has previously been dried and crushed. For the extraction, loweralcohols, preferably methanol or ethanol, are used. After evaporation ofthe solvent, a syrup remains behind which is subjected to distillationin a high vacuum in order to remove other highly effective oilysubstances. The solid distillation residue can be purified bychromatography on a column containing, for example, aluminum oxide orsilica gel. As eluant, organic solvents are used which contain,depending on their eluting capacity, 240% of a lower alcohol, preferablymethanol or ethanol. Suitable solvents are, for example, benzene,chloroform, methylene chloride, di-isopropyl ether, ethyl acetate,dioxane and tetrahydrofurane. The optimal mixing proportion of organicsolvent to alcohol can be determined by preliminary tests. Solventmixtures such, as chloroform/benzene in a ratio of 90:10 andbenzene/methanol in a ratio of 80:20 have worked Well.

The active substance crystallizes from the eluate as hydrochloride inthe form of weakly colored needles which can be purified byrecrystallization from ethanol. The free base may be obtained bytreating the hydrochloride with a base, for example with aqueousammonia. From the free base, other salts may be obtained, if desired, bytreatment with inorganic or organic acids, for example with sulfuricacid or acetic acid.

Prior to the extraction of the active substance, it is suitable toremove lipoid substances from the bark, for example, by extraction withpetroleum ether or carbon tetrachloride. It is also possible to remove,the above described active oils by extraction with chloroform prior tothe extraction with alcohols. This extraction should advantageosuly beelfected at room temperature, because, as described above, the substanceof the present invention is partially soluble in hot chloroform. Afterthe extraction United States Patent 0 3,655,886 Patented Apr. 11, 1972ice Percent C 62.7 H 6.5 N 4.1

A determination of the molecular weight by mass spectrometry showed amolecular weight of about 355. The substance shows a specific rotationof [a] =-+l72 (in ethanol); it has ultraviolet maxima at 270 ma (1 g. e=4.622) and 300 ma (1 g. e =4.249) (in methanol). The infrared spectrum(in KBr) is shown in the attached chart.

In thin-layer chromatography on silica gel, the substance has a R -valueof 0.36 using the system ethyl acetate/butanone/formic acid/water, inthe ratio of 5:3 1 1. It can be made visible on the chromatogram withDragendorffs reagent as an orange red spot.

The free base is soluble in methanol and chloroform. The hydrochlorideis easily soluble in lower alcohols such as methanol, ethanol, propanoland propylene glycol as Well as in Water, but is sparingly soluble inhot chloroform and hot acetatone, and is insoluble in petroleum ether,benzene, carbon tetrachloride, ether, tetrahydrofurane and dioxane. Withsilver nitrate, lead acetate, Reineoke salt, phosphotungstic acid andphosphomolybdic acid, the substance yields grey to brown coloredprecipitates.

The substance obtained according to the present invention isdistinguished by a strong and long lasting blood pressure loweringactivity. It provokes, when administered intravenously to a dog in adose of 0.5 mg./kg., a lowering of the blood pressure of 50 mm. lastingfor about 25 minutes. With 1.5 mg./kg., administered intravenously, itprovokes a lowering of the blood pressure of 75 mm., which is not yetcompensated after 30 minutes. The heart frequency is insignificantlylowered only temporarily. Respiration and electrocardiogram are notinfluenced.

The DL in a rat, upon intravenous administration, is about 5 mg./kg. ofbody weight.

Owing to its excellent blood pressure lowering activity, the newsubstance is generally suitable for the treatment of cardiac andcirculatory disorders, for example for the treatment of chronicalhypertonia, cardiac insufficiency, Angina pectoris and blood circulationdisorders. It can be administered perorally or intravenously. The dose,which depends on the weight of the patient and on the severity of thedisease, amounts to about 15-30 mg. in the case of intravenousadministration and to about 30-60 mg. in the case of oraladministration.

For oral administration, especially tablets or drages are used whichcontain the active substance in free form or in the form of a salt in aquantity of 5 to 30 mg. per dosage unit in addition to the usualadjuvants and carriers such as talc, starch, lactose, etc. Forintravenous administration, an aqueous solution of the substance ispreferably used.

The following examples illustrate the invention but they are notintended to limit it thereto:

EXAMPLE 1 465 g. of bark of Ravensara ar matica were extracted in aSoxhlet apparatus at first with petroleum ether and then, to exhaustion,with methanol. The methanol extract Was evaporated, whereafter a syrupybrown residue remained behind (29 g.). Oily substances were removed bydistillation at a bath temperature of 70 C. and a pressure of 1 mm. Hg.The solid residue of the distillation (19 g.) was adsorbed on g. ofaluminum oxide (Woelm neutral, activity degree I) and chromatographed on300 g. of aluminum oxide. Undesired accompanying substances were elutedwith chloroform. The active fraction could be eluted with chloroformcontaining 10% of methanol and crystallized from the eluate in weaklycolored needles. By recrystallization from ethanol, the substance wasobtained in the form of colorless and odorless needles. Yield? 78 mg.

EXAMPLE 2 529 g. of bark of Ravensara aromatica were stirred for 1 hourat room temperature with 2 liters of chloroform. The whole was thenfiltered and the operation was repeated twice. The bark was thenextracted in a Soxhlet apparatus with 96% ethanol. The residue (25 g.)obtained in dry state upon evaporation was adsorbed on g. of silica gel(Merck, grain size 0.2-0.5 mm.) and chromatographed on 500 g. of silicagel. The accompanying substances were eluted with benzene. The activefraction was eluted with a mixture of benzene and methanol andcrystallized from ethanol. Yield: 99 mg.

EXAMPLE 3 32 kg. of ground bark of Ravensara aromatica were extractedwith methanol in a large size extractor. The solution was concentratedunder reduced pressure to 5 liters. The concentrated product was stirredthree times each time with 3 liters of petroleum ether (65). The layerswere separated and the petroleum ether layer was rejected. The methanolphase was concentrated and the oils were removed-by distillation asdescribed in Example 1. The residue (197 g.) was adsorbed on 250 g. ofaluminum oxide (Woelm neutral, activity degree I) and chromatographed asdescribed in Example 1 on 5.5 kg. of aluminum oxide. The yield of activesubstance was 5.8 g., after recrystallization from ethanol.

We claim:

1. A process for preparing a pharmacologically active substance whichcomprises extracting the bark of Ravensara aromatica with methanol orethanol, evaporating the extract to dryness to give a solid residue, andthen separating the active substance from the residue by columnchromatography on aluminum oxide or silica gel using, as an eluant, amixture containing 2-3-0 percent of methanol or ethanol in an organiccarrier liquid in which said active substance is insoluble.

2. A process as in claim 1 wherein said bark is extracted with a memberselected from the group consisting of petroleum ether, chloroform, andcarbon tetrachloride prior to extraction with methanol or ethanol.

3. A pharmacologically active substance extracted from the bark ofRavensara ar mdtica according to the process of claim 1, said substancebeing soluble, as a free base, in lower alcohols, propylene glycol, andwater, and being insoluble in petroleum ether, benzene, carbontetrachloride, ether, tetrahydrofurane, and dioxane, and having thefollowing other physical properties:

Composition: carbon, hydrogen, oxygen, and nitrogen;

Elementary analysis (as free base): C, 62.7%; H,

Melting point: 223-224" 0.;

Molecular weight: about 355 (by mass spectrometry);

Ultraviolet absorption maxima (in methanol): 270,

300 millimicrons;

Ultraviolet absorption (in KBr) as in the accompanying Specificrotation: [u] =+172 (in ethanol);

R value=0.36 in ethyl acetate/'butanone/formic acid/ water (5:3:1z1).

4. A pharmaceutical composition for lowering blood pressure comprisingan effective amount of the pharmacologically active substance of claim 3in combination with a pharmaceutically acceptable carrier.

5. The method of lowering blood pressure which comprises orally orintravenously administering to a patient an effective amount of thepharmaceutically active substance of claim 3.

No references cited.

STANLEY J. FRIEDMAN, Primary Examiner U.S. Cl. X.R. 260236.5

